Strawberries (Fragaria X ananassa) are an important component of the U.S. specialty fruit crop. The fruit is grown in nearly every state, both commercially and in home gardens. The U.S. market value for strawberries was approximately $519 million in 1995, with a value of between $10,000 and $13,000 per acre harvested. Worldwide production is estimated at 2.4 million tons.
Strawberries are prone to multiple diseases including viruses, rots, leaf spots, and root and crown disease. Strawberry viruses are spread by aphids, nematodes, leafhoppers, and pollen. Viruses may also be transmitted from mother plants during planting. Pre- and post-harvest rotting of strawberries reduces yields by up to 15% annually. Gray mold caused by Botrytis cinerea is responsible for the majority of losses due to rot. Common leaf spot and leaf scorch reduce the vigor of infected leaves, lowering the robustness, yield, and quality of the fruit. Parasitic nematodes, bacteria, and fungi act to cause root and crown disease, lowering the yield of the crop.
In addition to traditional breeding techniques, incorporation of disease resistance, improvements to flavor and color, increased or modified sugar content, and other desirable traits can be envisioned using the modem tools of molecular biology.
Matthews, H. V. et al. (In Vitro Cell. Dev. Biol. 31: 36-43 (1995) and WO 95/35388, Dec. 28, 1995) describe the transformation of strawberries using Agrobacterium tumefaciens containing a binary vector. Explants of leaf, meristem, and petiole were co-cultured with Agrobacterium for 1-3 days, followed by an stepwise selection process in media containing 3% sucrose and increasing concentrations of kanamycin. All experimental plant cultures and tissues were maintained in a 16:8 light: dark photoperiod. Strawberry plants were regenerated from pure transgenic explants.
Nehra, N. S. et al. (J. Amer. Soc. Hort. Sci. 114: 1014-1018 (1989)) reported shoot regeneration from strawberry leaf disks. Efficient regeneration of Redcoat strawberries was achieved in media containing sucrose, benzyladenine, and indoleacetic acid. Low light intensities were found to be conducive to explant regeneration.
Nehra, N. S. et al. (Plant Cell Rep. 9: 10-13 (1990)) describe the transformation of strawberry via callus culture with Agrobacterium tumefaciens as the DNA delivery agent. Leaf explants were inoculated with bacterial suspension, and co-cultured on callus induction media containing 3% sucrose. Selection was performed for four weeks on media containing kanamycin, carbenicillin, and cefotaxime. Shoots were regenerated from selected calli, and rooted on medium containing kanamycin, benzyladenine, and indolebutyric acid.
James, D. J. and Barbara, D. J. (Acta Horticulturae 280: 495-502 (1990)) describe the transformation of apple and strawberry leaf disks and petioles. The method of Horsch, R. B. et al. (Science 227: 1229-1231 (1989), for tobacco and petunia) was used, varying the co-cultivation period, the type and quality of agar, plant growth regulators, and the length of the kanamycin selection step. All media used sucrose as a carbohydrate source.
James, D. J. et al. (Plant Science 69: 79-94 (1990)) describe low efficiency transformation of strawberry leaf disks with Agrobacterium tumefaciens. Plants were regenerated on MS medium supplemented with benzylaminopurine and 2,4-dichlorophenoxyacetic acid. Cefotaxime and kanamycin were used as selection agents, and sucrose was used as the carbohydrate source.
Nehra, N. S. et al. (Plant Cell Rep. 9: 293-298 (1990)) describes the transformation of strawberry via a leaf disk regeneration system. Leaf disks were inoculated with a non-oncogenic Agrobacterium tumefaciens strain harboring a binary vector plasmid. Disks were pre-cultured for 10 days on non-selective shoot regeneration medium containing 3% sucrose, and transferred to selective medium containing kanamycin. Selected shoots were multiplied on selective shoot proliferation media. Shoots were rooted and regenerated into strawberry plants.
Nyman, M. and Wallin, A. (Plant Cell Rep. 11: 105-108 (1992)) presented transient gene expression in strawberry protoplasts via electroporation. Purified protoplasts were suspended in an electroporation buffer containing 10 mM MES, 1 mM calcium chloride, and 0.5 M glucose as an osmotic support.
There exists a need for improved strawberry transformation methods to promote the engineering of desirable traits into this agronomically important crop.